The present invention is directed to glycosulfopeptides and methods of their use in treating inflammation and disorders related to leukocyte rolling mediated by P-selectin binding.
Inflammation is the reaction of vascularized tissue to local injury. This injury can have a variety of causes, including infections and direct physical injury. The inflammatory response can be considered beneficial, since without it, infections would go unchecked, wounds would never heal, and tissues and organs could be permanently damaged and death may ensue. However, the inflammatory response is also potentially harmful. Inflammation can generate pathology associated with rheumatoid arthritis, myocardial infarction, ischemia reperfusion injury, hypersensitivity reaction, and some types of fatal renal disease. The widespread problem of inflammatory diseases has fostered the development of many “anti-inflammatory” drugs. The ideal anti-inflammatory drug would be one that enhances the good effects resulting from the inflammatory response, and at the same time prevents or reduces the potentially harmful side-effects of this response.
The inflammatory response in regard to blood cells is accompanied by adhesion of circulating neutrophils, the most abundant phagocytic cell in the blood, to activated endothelial cells that line the vessels and make up the vessel walls. The adherent neutrophils are subsequently activated and the activated neutrophils emigrate from the blood into the surrounding tissue in a process termed diapedesis. The cells then begin engulfing microorganisms in a process termed phagocytosis and they also degranulate, releasing a variety of degradative enzymes, including proteolytic and oxidative enzymes into the surrounding extracellular environment. The mechanisms by which neutrophils adhere, become activated, and emigrate from the blood are currently major topics of research around the world.
Leukocyte recruitment to inflamed tissues is a highly ordered process that begins with and is to a large extent reliant on selectin-dependent leukocyte rolling. Inhibiting selectin binding therefore holds great promise for the treatment of inflammatory diseases and conditions. The selectin family of adhesion molecules has three functionally and structurally related members, namely E-selectin (expressed by endothelial cells) L-selectin (expressed by leukocytes) and P-selectin (expressed by endothelial cells and platelets). P-selectin has been convincingly implicated in inflammatory disorders including ischemia-reperfusion injury and atherosclerosis. Leukocyte rolling is supported by rapid formation of selectin-selectin ligand bonds at the front of a cell, coupled with detachment at the rear. With a constant requirement for new bond formation, leukocyte rolling is therefore sensitive to treatments that block the molecules involved in this response. In keeping with this model, application of antibodies that block the selectins or PSGL-1 (P-selectin glycoprotein ligand-1) should cause reversal of existing leukocyte rolling in vivo. Charged polysaccharides such as fucoidin and dextran sulfate can also inhibit preexisting leukocyte rolling, presumably by binding to and blocking the selecting.
The realization that the selectin family of adhesion molecules all recognize sialylated fucosylated glycans, prototypically represented by the tetrasaccharide sialyl Lewisx (sLex), fueled development of carbohydrate based selectin inhibitors. Data from in vitro binding assays and from models of inflammation support the notion that sLex-mimetic drugs inhibit all three selectins and, as such, should be efficacious against inflammatory disease. Using an intravital microscopy model, where leukocyte rolling was observed immediately before and after application of inhibitors, it was shown that sLex and close structural mimetics thereof are, in fact, weak inhibitors of E-selectin dependent rolling and have no impact whatsoever on P- or L-selectin dependent rolling. This fact is consistent with the notion that sLex and related structures represent only one component of the macromolecular assemblies that represent true selectin ligands.
The best characterized selectin ligand is PSGL-1, a dimeric mucin present on all leukocytes. Studies with antibodies and with gene-targeted mice lacking PSGL-1 have demonstrated that PSGL-1 is the major ligand for P-selectin dependent leukocyte rolling in the microcirculation. In addition, it was demonstrated that recombinant PSGL-1 fused to human IgG (rPSGL-Ig) could support rolling interactions of microspheres with E- and P-selectins in venules and could competitively inhibit leukocyte rolling on E- and L- as well as P-selectin in vivo. However, difficulties of large scale synthesis and fears of immune reactions limit the use of antibodies for therapy, whereas a high possibility of nonspecific side effects limit the use of fucoidin and similar agents. Therefore, smaller molecules of defined structure that selectively bind to selectins with high affinity and which prevent binding of selectins to ligands could comprise attractive drug candidates.